Proteolysis restricts localization of CID, the centromere-specific histone H3 variant of Drosophila, to centromeres

Abstract

Centromere identity is determined by the formation of a specialized chromatin structure containing the centromere-specific histone H3 variant CENP-A. The precise molecular mechanism(s) accounting for the specific deposition of CENP-A at centromeres are still poorly understood. Centromeric deposition of CENP-A, which is independent of DNA replication, might involve specific chromatin assembly complexes and/or specific interactions with kinetochore components. However, transiently expressed CENP-A incorporates throughout chromatin indicating that CENP-A nucleosomes can also be promiscuously deposited during DNA replication. Therefore, additional mechanisms must exist to prevent deposition of CENP-A nucleosomes during replication and/or to remove them afterwards. Here, using transient expression experiments performed in Drosophila Kc cells, we show that proteasome-mediated degradation restricts localization of Drosophila CENP-A (CID) to centromeres by eliminating mislocalized CID as well as by regulating available CID levels. Regulating available CID levels appears essential to ensure centromeric deposition of transiently expressed CID as, when expression is increased in the presence of proteasome inhibitors, newly synthesized CID mislocalizes. Mislocalization of CID affects cell cycle progression as a high percentage of cells showing mislocalized CID are reactive against alphaPSer(10)H3 antibodies, enter mitosis at a very low frequency and show strong segregation defects. However, cells showing reduced amounts of mislocalized CID show normal cell cycle progression.

Figure 1

Transiently expressed CID-YFP shows two coexisting patterns of localization, at centromeres and throughout chromatin. CID-YFP (A), HFD_CID-YFP (B) and N_H3HFD_CID-YFP (C) can localize throughout chromatin (a), only at centromeres (c) and both, at centromeres and throughout chromatin (b). N_CIDHFD_H3-YFP (D) localizes only throughout chromatin. YFP is shown in green. DAPI-staining is shown in blue. The immunolocalization pattern obtained with αN_CID, which specifically recognizes the N-terminal domain of CID, is shown in red only in (B). Bars correspond to 5 μm, except in row c of (B) where it corresponds to 10 μm.

Figure 2

Upon cell culture, CID-YFP localization is constrained to centromeres. (A) The percentage of total transfected cells showing CID-YFP mislocalized throughout chromatin (white columns), localized exclusively at centromeres (black columns) or both, at centromeres and throughout chromatin, (grey columns) are presented as a function of increasing time after transfection. Results are the average of three independent experiments. (B) As in (A) but for HFD_CID-YFP. In (C), cells showing mislocalized CID-YFP were sorted by FACS 48 h after transfection and cultured for an additional 24 h. The percentage of cells showing mislocalized CID-YFP (white columns), CID-YFP localized only at centromeres (black columns) or a mixed localization pattern (grey columns) is presented as a function of increasing time after sorting. Results are the average of three independent experiments. In (D), the levels of CID-YFP mRNA are analyzed by RT–PCR at different times after transfection using primers specific for YFP (bands labelled YFP) and, as a control, primers specific for actin5C (bands labelled act). Kc corresponds to untransfected cells. Reactions were performed with 0.5 ng (lane 1) and 5 ng (lane 2) of total RNA. As a negative control, the results of similar reactions where the two sets of primers were added after the RT-step but before PCR-amplification are shown at the bottom.

Figure 3

Clearance of mislocalized CID-YFP does not take place at any given time during cell cycle progression. (A) Time-lapse analysis of the process of clearance in interphase nuclei is presented as a function of increasing time of visualization. YFP is shown in yellow (a) and merged to transmission (b). (B) Condensed metaphase chromosomes showing CID localized exclusively at centromeres (a) and both, at centromeres and throughout chromatin (b). YFP is shown in green. DAPI-staining is shown in blue. Bars correspond to 5 μm.

Figure 4

Centromeric localization of transiently expressed CID-YFP is perturbed in the presence of the proteasome inhibitor MG132. (A) The percentage of total transfected cells showing CID-YFP localized exclusively at centromeres 48 h after transfection are presented as a function of increasing time of treatment with 5 μg/ml of MG132: no treatment (white columns), 3 h (dotted columns), 6 h (grey column) and 9 h (black columns). Results are the average of three independent experiments. In (B), cells showing mislocalized CID-YFP were sorted by FACS 48 h after transfection and cultured for an additional 24 h either in the presence of 5 μg/ml of MG132 or in the absence of any added inhibitor. The percentage of cells showing CID-YFP localized exclusively at centromeres is presented immediately after sorting (white column) and after 24 h of additional culture in the presence of MG132 (grey column) or in its absence (black column). Results are the average of three independent experiments. (C) The percentage of total transfected cells that, 48 h post-transfection, show CID-YFP exclusively localized at centromeres after treatment with 5 μg/ml of MG132 for 3 h, 6 h and 9 h (black columns) is compared with the percentage of cells showing CID-YFP localized exclusively at centromeres at the time when the inhibitor was added (white columns). Results are the average of three independent experiments.

Figure 5

Cell cycle progression is altered in cells showing mislocalized CID-YFP. (A) Cells were stained with αPSer¹⁰ 48 h after transfection of CID-YFP. YFP is shown in green. αPSer¹⁰ is shown in red. DAPI-staining is shown in blue. Bar corresponds to 5 μm. (B) The percentage of cells that 48 h after transfection of CID-YFP, HFD_CID-YFP and N_CIDHFD_H3-YFP are positive to αPSer¹⁰ are presented as a function of the pattern of localization: untransfected cells (white columns); cells showing CID-YFP localized exclusively at centromeres (dotted columns); cells showing mislocalized CID-YFP (black columns) and cells showing CID-YFP at centromeres and throughout chromatin (dashed columns). Results are the average of three independent experiments.

Figure 6

Cells showing a mixed pattern of CID-YFP localization, at centromeres and throughout chromatin, progress normally through mitosis. Time-lapse analysis of cells showing mislocalized CID-YFP (B) or a mixed pattern of CID-YFP localization (A) are presented as a function of increasing time of visualization. YFP is shown in yellow (a) and merged to transmission (b). Bars correspond to 5 μm.